Preparations consisting of the cell walls of Mycobacterium bovis or M. smegmatis possess potent activity in experimental as well as human cancer immunotherapy. Our long-term objective is to identify, isolate, structurally characterize, and, where appropriate, synthesize the components of these cell walls that are requisite for immunotherapeutic activity. Since macrophage activation is known to be important in this process, we are examining at a chemical level the processing (degradation) of bacterial cell walls by macrophages. The rationale for doing this is that a small fragment of bacterial peptidoglycan, muramyl dipeptide, has previously been shown to function as an immunostimulant and to act upon the macrophage. These facts suggested to us a mechanism for macrophage involvement in the recognition of bacterial infection and in immune amplification. The hypothesis underlying our work is that the macrophage would phagocytize and catabolize an invading bacterium, releasing glycopeptides unique to bacteria to serve as signals for immune amplification. The processing of bacterial cell walls by macrophages is being examined by feeding Bacillus subtilis cell walls radiolabeled in the amino sugar and amino acid residues of peptidoglycan to a murine macrophage cell line (RAW264) grown in culture. The radiolabeled glycopeptides that are excreted have been isolated, characterized, and analyzed as macrophage activators. During the course of this work we also made the observation that one of these peptidoglycan-derived glycopeptides was being retained by the macrophage and converted to a covalent lipid derivative ("peptidoglycolipid"). Our initial studies strongly implicate this peptidoglycolipid as being the agent responsible for the intense blastogenic activity associated with the phospholipid fraction derived from macrophages exposed to bacterial cell walls. The purification and structural characterization of this component will be the focus of this work. We also propose to analyze its effects on immune functions to include macrophage tumoricidal and bacteriacidal activities and lymphocyte blastogenesis and antibody production. The long term goal of this research is to identify adjuvant-active compounds that can be used to potentiate macrophage and lymphocyte function in order to enhance immunological control of malignancy and microbial infection.